The present invention relates to an expression construct which is capable of producing high levels of erythropoietin in mammalian cells. More particularly, the present invention relates to an expression construct which includes an erythropoietin coding region fused to a unique 5′-UTR sequence and to the use of such construct for synthesizing large amounts of erythropoietin and in increasing serum erythropoietin level in individuals in need thereof.
Erythropoietin (EPO) is produced in the adult kidney or the fetal liver in response to tissue hypoxia. Thus, a reduction of tissue oxygen level results in upregulation of the EPO gene and in increased levels of EPO in the serum. Increased levels of EPO inhibit apoptosis of the erythroid progenitor cell in the bone marrow and stimulate its proliferation and differentiation which result in a release of erythrocytes into the blood stream [Krystal, G. (1983) Exp. Hematol. 11, 649-660; Powell J S. et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6465-6469]. On the other hand, lack of EPO in the serum would lead to anemia with fatigue and cellular hypoxia.
Several clinical conditions such as anemia, lung disease or cyanotic heart disease give rise to tissue hypoxia which leads to increased levels of serum EPO. However, in patients with renal insufficiency, serum EPO levels remain low in spite of hypoxia. In addition, abnormally low levels of serum EPO are also seen in anemic patients suffering from cancer, rheumatoid arthritis, HIV infection, ulcerative colitis, sickle cell anemia and anemia of prematurity.
While the causes of abnormally low levels of EPO include a primary defect in EPO production in cases of renal disease. and anemia of prematurity, and a suppression of EPO synthesis by inflammatory cytokines (e.g., IL-1, TNF-α) in cases of certain chronic diseases and cancer, administration of exogenous EPO is expected to circumvent the low levels of EPO in such conditions.
Indeed, recombinant human EPO (Rhu-EPO) has been successfully used in a variety of clinical conditions to increase production of red blood cells. Currently, erythropoietin is licensed for use in the treatment of anemia of renal failure, anemia associated with HIV infection in zidovudine (AZT) treated patients, anemia associated with cancer chemotherapy, myelodysplastic syndromes, prematurity, autologous blood donation and bone marrow transplantation [Henry, D H. and Spivak, J L. (1995). Curr. Opin. Hematol. 2: 118-124].
However, while EPO administration can benefit a wide variety of patients its use as a therapeutic agent is limited due to low production yields and high production costs.
EPO has been produced from expression vectors using transient and stable transfections in mammalian cells. These expression vectors included a 3.3 kb EPO cDNA under the control of the adenovirus major late promoter [Jacobs K et al. (1985) Nature 313, 806-809], a 5.4 kb long genomic EPO (gEPO) containing the 5′ and 3′ flanking regions transcribed from the simian virus 40 (SV40) promoter [Lin F K et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7580-7584], a 2.4 kb ApaI fragment of the EPO gene containing 58 bp of the 5′ UTR (U.S. Pat. No. 5,688,679 to Powell, J. S., 1986), and a 5′ and 3′ UTR-deleted EPO genomic clone (Park, J. H. et al., Biotechnol. Appl. Biochem. (2000) 32: 167-72). However, these transfections resulted in relatively low yields of EPO, which varied between 2-7 IU/ml EPO in 48 hours when the transcribed sequence included 5′ and 3′ flanking regions, to 56-68 IU/ml in 48 hours when truncated 5′-UTR or UTR-deficient EPO constructs were utilized.
There is thus a widely recognized need for, and it would be highly advantageous to have, an expression system capable of producing high levels of EPO devoid of the above limitations.